The correct balance between excitation and inhibition (E/I balance) in neuronal circuits is essential for learning and memory, cognition and behavior. Disrupted inhibition leading to elevated neuronal and circuit excitability is thought to underlie the pathology of numerous neurological disorders, therefore a comprehensive understanding of the molecular mechanisms involved in synaptic inhibition will have the potential to direct new therapeutic strategies for treating these conditions. GABAergic inhibitory synapses mediate the majority of synaptic inhibition in the central nervous system, and their plasticity controls neuronal excitability and function. The number of GABAA receptors (GABAARs) at inhibitory postsynaptic sites is a key determinant of inhibitory synapse strength and hence neuronal inhibition. Therefore defining mechanisms by which synaptic GABAARs are clustered and how they are altered in synaptic plasticity is imperative for understanding inhibition and its disruption in brain disorders. Using the versatile super-resolution imaging technique, Structured Illumination Microscopy (SIM), we find that GABAARs and their scaffold, gephyrin, form nanoscale subsynaptic domains (SSDs) in the inhibitory postsynaptic domain, are modulated during plasticity, and form closely associated pairs with presynaptic release SSDs in the active zone, suggesting that a modular nanoscale architecture for the inhibitory synapse may be important for their plasticity and function. Our goal is to determine the mechanisms that control inhibitory nanoscale organization, understand how this organization influences inhibitory synaptic function and is altered during synaptic plasticity, and determine how these facets differ between diverse inhibitory synapse subtypes. This proposal will (1) determine the mechanisms underlying the formation of postsynaptic inhibitory SSDs during activity-dependent synapse growth, (2) define the mechanisms and functional relevance of postsynaptic SSD clustering opposite presynaptic release sites, and (3) examine how subcellular location and interneuron input influence inhibitory nanoscale organization. These aims will test our overarching hypothesis that inhibitory synaptic nanoscale organization underlies inhibitory synaptic strength, and its dynamic regulation is a crucial mechanism for synaptic plasticity. These proposed studies will be significant, being the first comprehensive mechanistic and functional examination of inhibitory synapse architecture at nanoscale resolution (in both culture and slice) and in real-time during short- and long-term plasticity paradigms. The widespread importance of this work is that it will greatly expand our understanding of the detailed mechanisms that control inhibitory synaptic plasticity and inhibition, which is critical for maintaining E/I balance and neuronal function. Moreover, determining the nanoscale structure of inhibitory synapses in healthy brains will pave the way for future studies in disease models to provide novel understanding of how inhibitory synapse structure, function and E/I balance are disrupted in neuropathological disorders.